RNF126-Mediated Reubiquitination Is Required for Proteasomal Degradation of p97-Extracted Membrane Proteins.

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.

Metabolic engineering of Escherichia coli W3110 to produce L-malate.

A four-carbon dicarboxylic acid L-malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L-malate production via one-step L-malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), pyruvate formate lyase (pflB), phosphotransacetylase (pta), and acetate kinase A (ackA) in pta-ackA pathway led to accumulate 20.9 g/L pyruvate. Then, overexpression of NADP+ -dependent malic enzyme C490S mutant in this multi-deletion mutant resulted in the direct conversion of pyruvate into L-malate (3.62 g/L). Next, deletion of the genes responsible for succinate biosynthesis further enhanced L-malate production up to 7.78 g/L. Finally, L-malate production was elevated to 21.65 g/L with the L-malate yield to 0.36 g/g in a 5 L bioreactor by overexpressing the pos5 gene encoding NADH kinase in the engineered E. coli F0931 strain. This study demonstrates the potential utility of one-step pathway for efficient L-malate production. Biotechnol. Bioeng. 2017;114: 656-664. © 2016 Wiley Periodicals, Inc.

Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production.

L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L-1, 0.30 g g-1, and 0.32 g L-1 h-1 in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L-1, 0.31 g g-1, and 0.32 g L-1 h-1, respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.

Mitochondrial engineering of the TCA cycle for fumarate production.

Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, mitochondrial engineering was used to construct the oxidative pathway for fumarate production starting from the TCA cycle intermediate α-ketoglutarate in Candida glabrata. Accordingly, α-ketoglutarate dehydrogenase complex (KGD), succinyl-CoA synthetase (SUCLG), and succinate dehydrogenase (SDH) were selected to be manipulated for strengthening the oxidative pathway, and the engineered strain T.G-K-S-S exhibited increased fumarate biosynthesis (1.81 g L(-1)). To further improve fumarate production, the oxidative route was optimized. First, three fusion proteins KGD2-SUCLG2, SUCLG2-SDH1 and KGD2-SDH1 were constructed, and KGD2-SUCLG2 led to improved fumarate production (4.24 g L(-1)). In addition, various strengths of KGD2-SUCLG2 and SDH1 expression cassettes were designed by combinations of promoter strengths and copy numbers, resulting in a large increase in fumarate production (from 4.24 g L(-1) to 8.24 g L(-1)). Then, through determining intracellular amino acids and its related gene expression levels, argininosuccinate lyase in the urea cycle was identified as the key factor for restricting higher fumarate production. Correspondingly, after overexpression of it, the fumarate production was further increased to 9.96 g L(-1). Next, two dicarboxylic acids transporters facilitated an improvement of fumarate production, and, as a result, the final strain T.G-KS(H)-S(M)-A-2S reached fumarate titer of 15.76 g L(-1). This strategy described here paves the way to the development of an efficient pathway for microbial production of fumarate.

Immunohistochemical expression of cytokeratins in human salivary gland acinic cell carcinomas.

OBJECTIVE: To compare the expression of cytokeratins (CKs) in the solid, microcystic, follicular, and papillary-cystic subtypes of salivary gland acinic cell carcinoma (AcCC) in order to characterize the cell origin. STUDY DESIGN: The expression of CK7, CK14, CK19, CK20, and alpha-smooth muscle actin (α-SMA) in 18 cases of AcCC was assessed with the use of immunohistochemical staining. Ten normal salivary glands were used as controls. RESULTS: The expression of CKs in AcCCs varied according to their growth patterns. CK7 showed strong and diffuse positive staining in the microcystic, follicular, and papillary-cystic subtypes, whereas staining was weakly positive or negative in the solid subtype. CK14 expression was negative in almost all AcCCs. Expression of CK19 was observed in the microcystic, follicular, and papillary-cystic subtypes, but was minimally observed in the solid subtype. No cells positive for CK20 or α-SMA were found in any AcCCs. CONCLUSIONS: We demonstrated that the microcystic, follicular and papillary-cystic subtypes of AcCC exhibit features of ductal luminal cells with expression of CK7 and CK19, suggesting their ductal origination. By contrast, the solid subtype might originate from different cells with no ductal CK expression.

Enzymatic production of α-ketoglutaric acid from l-glutamic acid via l-glutamate oxidase.

In this study, a novel strategy for α-ketoglutaric acid (α-KG) production from l-glutamic acid using recombinant l-glutamate oxidase (LGOX) was developed. First, by analyzing the molecular structure characteristics of l-glutamic acid and α-KG, LGOX was found to be the best catalyst for oxidizing the amino group of l-glutamic acid to a ketonic group without the need for exogenous cofactor. Then the LGOX gene was expressed in Escherichia coli BL21 (DE3) in a soluble and active form, and the recombinant LGOX activity reached to a maximum value of 0.59U/mL at pH 6.5, 30°C. Finally, the maximum α-KG concentration reached 104.7g/L from 110g/L l-glutamic acid in 24h, under the following optimum conditions: 1.5U/mL LGOX, 250U/mL catalase, 3mM MnCl2, 30°C, and pH 6.5.