Peer review of: Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and (2020)

Reviewer #1 (Remarks to the Author): 

Ma and colleagues engineered ectoine overproduction in Halomonas bluephagenesis with/without 
polyhydroxyalkanoate overproduction. Halomonas synthesizes ectoine upon hyperosmotic stress. 
Overexpression of the endogenous ectABC operon allowed for ectoine overproduction, deletion of 
ectoine converting genes (doeA, ectD) and overexpression of precursor genes improved production. 
Open bioreactors were used to produce 28 g/L ectoine. As expected, nitrogen limitation interfered with 
ctoine production which was reduced to 8 g/L since carbon was lost for polyhydroxybutyrate 
production. 
Major criticism: 
Conceptionally, it is not clear why the author stress coproduction? Is ectoine to be used in bioplastics? 
What would be an advantage of ectoine + PHB coproduction as compared to individual production of 
ectoine and PHB at much higher levels and mixing separately produced PHB and ectoine? 
Bioreactor data are from a preliminary strain (TD1.0 according to the caption). Authors improved this 
strain (s. strain list TD-ADE-58). Why have they not compare TD1.0 with TD-ADE-58? These data 
should be shown. 
Authors may delete genes for PHB biosynthesis to boost ectoine production. 
To deduce the function of the conserved ectABC encoded proteins, authors may construct and analyse 
deletion mutants. 

Authors shall calculate ectoine yields on glucose and volumetric productivities. 

All figure legends: were replicate cultivations analysed or sample quantification was done in replicates: 
explain here 
Figure 7 legend should contain information how many biological replicates were analysed and explain 
meaning of error bars. If no replicates were performed indicate this fact. 

Suppl table: 
List of genes: give gene IDs from Genbank instead of a ref. or “this study” 

Reviewer #2 (Remarks to the Author): 

In the manuscript, the authors build on their previous work with a halophilic bacterium, engineering it 
to produce high concentrations of ectoine, a solute produced by this class of organisms. The authors 
verify that their chosen organism, Halomonas bluephagenesis, contains the ectoine biosynthetic 
pathway, and perform a number of genetic manipulations along with medium formulation to achieve a 
maximum titer of 28 g/L in an “open” fermentation. Lastly, they demonstration the co-production of 
ectoine with the biopolymer PHB, albeit at lower titers. The manuscript is relatively straightforward, 
though some parts can be a bit confusing. The titers achieved are impressive, although the methods 
employed have all been previously reported. The following specific comments are intended to help 
strengthen the manuscript: 

1. Line 54 – I do not believe it is fair to call a sterile bioprocess “complicated” as it implies that this is 
not the current standard in the biotech industry. Non-sterile processes can also be “complicated.” 

2. Lines 154-156 – The medium named “60MME” has 60 g/L NaCl by definition. Therefore, why was 
there a “factorial analysis” to find the optimized concentration? 

3. Line 157, Figure 3c – 60 g/L was noted to give the “highest” concentration at 2.82 g/L. Is this really 
significantly different from what was achieved with 80 g/L? 

4. Lines 165-167 – It should be noted that the value of 2.83 g/L achieved with urea matches but 

doesn’t exceed that obtained with aspartate supplementation. As written, it is easy for the reader to 
mistake this as a significant improvement. 

5. Lines 212-214 – What was the “stronger promoter” that could not be constructed? 

6. Line 221 – It is not “remarkable” that the negative control strain is so low – that’s why it is the 
negative control! 

7. Line 232 – I don’t agree with the use of the word “biosensor” here. Biosensors respond to specific 
metabolites or ligands and regulate gene expression in response. These are simply chemically-
inducible promoters, which are both very useful and well-established. 

8. Line 253 – Most “induction combinatory groups had enhanced production” over what? Only the 
induced results are shown in Figure 6f, so it’s not clear what the reference point should be. 

9. Line 259-262 – The first two sentences are a bit confusing. I think it would help to include the 
promoter that *did* work for lysC. As written, the reader could be left thinking that lysC was not 
overexpressed at all, only asd. 

10. Line 262 – I think “6h” should be “6g” since there is no panel “6h”. 

11. Line 328 – Please use “quorum-sensing-based” rather than “sensor-based” since this is more 
descriptive. 

12. Line 342 – The authors are free to ignore this suggestion, but I would note that PHB is derived 
from acetyl-CoA rather than acetoacetyl-CoA. It is one step further back, but is a much more well-
known metabolic intermediate. 

13. Methods – In the initial description of the medium, yeast extract is listed as an ingredient. In 
many places that follow, this becomes just “yeast” which I assume is a mistake. 

14. Figure 2a – The heat map with black is very hard to read. Since the rest of the figure is in color, I 
strongly suggest using a color scale to represent the data. 

15. Figure 3a – A very short description of the “T7-like” system, even just specifying what MmP1 is, 
would be helpful. 

16. Figure 4, lines 57-59 – The description of the strain names makes it seem like TD1.0 does NOT 
contain the ectoine synthesis plasmid. Is this correct? 

17. Figure 6, panel g legend – The order of 60MMU and 60MMU5 in the legend should match the order 
the data are presented (i.e., 5 g/L of urea is first in the figure). 

18. Figure S7 is not referenced anywhere in the main text. 

Language errors (these are just a few – please proofread carefully): 

19. Lines 76-79 – This sentence does not read quite right. Perhaps it should say “…were successfully 
used to direct metabolic fluxes…”? 

20. Line 88 – “engineering” should be “engineer” 

21. Line 98 – “depended” should be “dependent” 

22. Line 120 – “heterogeneous” should be “heterologous” (and in other places, too) 

23. Line 120 – ectoine is misspelled 

24. Line 155 – “mediums” should be “media” 

25. Line 179 – “double checked” should be “confirmed” 

26. Line 199 – I don’t think “variable” is the right word – “reporter” is more standard. 

27. Line 262 – Is “also” correct or should this be “almost”? 

28. Lines 268-270 – If I am reading Fig S4 correctly, the 96-well plate cultures were done. That 
means “should be” should be “were”. 

29. Line 397 – “Qiagen” is misspelled 

30. Line 456 – “whatever” should be “whenever” 

31. Figure S4, line 77 – remove “respectively” 

32. Figure S5, line 90 – “guildance” should be “guidance” 

Reviewer #3 (Remarks to the Author): 

Dear Authors, 

I reviewed your highly intriguing manuscript, which constitutes the next elaboration on Halomonas 
bluephagenesis, the prototype PHA production strain used for PHA production in the context of “Next 
Generation Industrial Biotechnology” (NGIB), which comprises the cultivation of engineered robust, 
powerful production strains under minor sterility precautions in a cost-efficient manner. 
The manuscript at hand deals with the co-production of PHA (homopolyester PHB) and the osmo-
solute ectoine; while PHB accrues as intracellular product, ectoine can be harvested as extracellular 
product via “bacterial milking” in hyposaline medium. 
A new ectoine biosynthesis pathway was constructed in H. bluephagenesis by fine-tuning the 
transcription levels of involved genes using two biosensor responsive systems. In fed-batch 
cultivation, the genetically engineered bacterial construct was able to produce a total of 8 g/L and 32 
g/L cell dry mass harboring 75 wt.-% PHB within 28h of cultivation. 
Indeed, the work contributes to the development of versatile microbial “working horses”, which are 
capable of co-producing different marketable bioproducts under inexpensive cultivation conditions, 
which is the pre-condition for industrial-scale feasibility. 
The work is scientifically sound, the experiments are well designed and reproducible for peers, and the 
conclusions are well supported by the elaborated data. For sure, the work is of interest for the 
scientific community active in this field. 
Only some minors should be addressed in order to further improve the submission: 
A) Abstract: no need to abbreviate the species name in brackets at first occurrence (line 30-31) 
B) Title: “Rational Flux-Tuning Halomonas bluephagenesis for…” should rather be “Rational Flux-
Tuning of Halomonas bluephagenesis for…” 
C) Polymer name: “polyhydroxybutyrate” should be “poly(3-hydroxybutyrate)” (think about poly(4-
hydroxybutyrate)”. In abstract and text. 
D) Some English editing will welcome, e.g., line 46: “the most well-known”: better: “the best known”; 
line 51: “…, organ…”: “…, and organ…”; line 69: “saltless”: better: desalted”; line 88: “to engineering 

H. bluephagenesis”: “to engineer H. bluephagenesis”; line 102: “economy improvement”: “economic 
improvement”; line 116-117: “..blast results…shows…”: “..blast results…show…”; line 274: “Ectoine”: 
“ectoine”; line 328: “It has to be note”: “It has to be noted”. Line 495: “Follwed by…..quantification”: 
please, rephrase this sentence; it is not clear. 
E) A minor: “L-aspartate” etc.: “L” should not be in capital letter, but, scientifically correct, in small 
caps. 
F) Check style of references in text: e.g., line 50: “2, 3, 4, 5, 6, 7”: I suppose, it should be “2-7” (also 
elsewhere in text) 
G) Line 85: “by man bacteria”: add “and archaea”; not only bacteria accumulate PHA! 
H) Figure legend 1 (line 10) and elsewhere: “β-ketothiolase” today is “3-ketothiolase”; Figure 1: 
Check abbreviation: “P3HB” is “PHB” in the main text and legend! Line 9 in legend: “and” should not 
be in italics