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Ko, Yeounjoo,Seol, Eunhee,Sundara Sekar, Balaji,Kwon, Seongjin,Lee, Jaehyeon,Park, Sunghoon Elsevier 2017 Bioresource technology Vol.244 No.1
<P><B>Abstract</B></P> <P>Production of 3-hydroxypropionic acid (3-HP) or 1,3-propanediol (1,3-PDO) production from glycerol is challenging due to the problems associated with cofactor regeneration, coenzyme B<SUB>12</SUB> synthesis, and the instability of pathway enzymes. To address these complications, simultaneous production of 3-HP and 1,3-PDO, instead of individual production of each compound, was attempted. With over-expression of an aldehyde dehydrogenase, recombinant <I>Klebsiella pneumoniae</I> could co-produce 3-HP and 1,3-PDO successfully. However, the production level was unsatisfactory due to excessive accumulation of many by-products, especially acetate. To reduce acetate production, we attempted; (i) reduction of glycerol assimilation through the glycolytic pathway, (ii) increase of glycerol flow towards co-production, and (iii) variation of aeration rate. These efforts were partially beneficial in reducing acetate and improving co-production: 21g/L of 1,3-PDO and 43g/L of 3-HP were obtained. Excessive acetate (>150mM) was still produced at the end of bioreactor runs, and limited co-production efficiency.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>K. pneumoniae</I> was engineered for co-production of 3-HP and 1,3-PDO from glycerol. </LI> <LI> Co-production improved upon by-products reduction. </LI> <LI> The maximum 21g/L 1,3-PDO and 43g/L 3-HP were obtained in fed-batch bioreactor cultivation. </LI> <LI> Acetate accumulation was serious and limited further improvement of co-production. </LI> <LI> Activation of TCA cycle and ETC is suggested to reduce acetate and improve ATP supply. </LI> </UL> </P>
새로이 분리된 Klebsiella pneumoniae 균주들의 글리세롤 기반 3-hydroxypropionic acid 및 1,3-propanediol 동시 생산성 평가
고연주(Yeounjoo Ko),설은희(Eunhee Seol),순달아 세칼 바라지(Balaji Sundara Sekar),권성진(Seongjin Kwon),이재현(Jaehyeon Lee),박성훈(Sunghoon Park) 한국생물공학회 2016 KSBB Journal Vol.31 No.4
Co-production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) was suggested as an innovative strategy to overcome several limitations occurring in the single production of 3-HP from glycerol. In this study, two new isolates of Klebsiella pneumoniae, which produce less lipopolysaccharide (LPS) thus considered less pathogenic than K. pneumoniae DSM 2026, were compared and evaluated for their potential for the co-production of 3-HP and 1,3-PDO. The newly isolated strains showed significantly faster sedimentation rate than DSM, which should be beneficial for downstream processing. Analysis of genome sequences of the isolates confirmed the presence of all genes necessary for glycerol assimilation, 1,3-PDO production and biosynthesis of coenzyme B12. Co-production yield was highest under anaerobic condition while cell growth was highest under aerobic condition. Both strains showed similarly good performance for the co-production although J2B gave the slightly higher co-production yield of 0.80 mol/mol than GSC021 (0.75 mol/mol). The evaluation of the newly developed strains presented here should be useful in designing similar evaluation experiments for other microorganisms.
Ashok, Somasundar,Sankaranarayanan, Mugesh,Ko, Yeounjoo,Jae, Kyeung‐,Eun,Ainala, Satish Kumar,Kumar, Vinod,Park, Sunghoon Wiley Subscription Services, Inc., A Wiley Company 2013 Biotechnology and bioengineering Vol.110 No.2
<P><B>Abstract</B></P><P>3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant <I>Escherichia coli</I> stains can produce 3‐HP from glycerol in the presence of vitamin B<SUB>12</SUB> (coenzyme B<SUB>12</SUB>), when overexpressed with a coenzyme B<SUB>12</SUB>‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant <I>Klebsiella pneumoniae</I> strains, which naturally synthesizes vitamin B<SUB>12</SUB> and does not require supplementation of the expensive vitamin. The NAD<SUP>+</SUP>‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of <I>K. pneumoniae</I> alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B<SUB>12</SUB> addition, the recombinant <I>K. pneumoniae</I> Δ<I>dhaT</I>Δ<I>yqhD</I> overexpressing PuuC could produce ∼3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD<SUP>+</SUP> regeneration (under low aeration) or reduced vitamin B<SUB>12</SUB> synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, <I>K. pneumoniae</I> Δ<I>dhaT</I>Δ<I>yqhD</I>, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that <I>K. pneumoniae</I> is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B<SUB>12</SUB>. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.</P>